Animal feed composition for prevention of mycotoxicoses and method for preparing same



United States Patent 3,255,014 A IMAL FEED COMPOSITION FOR PREVENTEON23F MYCOTOXICOSES AND METHOD FOR PRE- PARING SAME Joseph F orgacs, PearlRiver, N.Y., assignor to American Cyanamid Company, Stamford, Conn, acorporation of Maine No Drawing. Filed Jan. 23, 1963, Ser. No. 253,282 2Gaims. (Cl. 99-4) This application is a 'continuation-in-part of mycopending application Serial No. 850,075, filed :November 2, 1959, nowabandoned, which is a continuatlon-m-part of my application Serial No.582,323, filed May 3, 1956, now abandoned. This invention relates tocereal compositions wherein the elaboration of fungal metabolicbyproducts, which are toxic to animals ingesting the same, is inhibited.Furthermore, the invention is adapted to animal feed compositionscontaining less than fung1cidal concentraitons of 8-hydroxyquinoline,for the prevention of mycotoxicoses in domestic livestock and otheranimals.

Poultry hermorrhagic syndrome is an example of a mycotoxicoses and ischaracterized by certain abnormal clinical symptoms and by hemorrhagesin the legs, thighs and other organs, as well as by degenerative changesin the liver and kidneys, and by a pronounced depression of theblood-forming tissues.

Although death losses as a result of the hemorrhagic syndrome range from0 to over 50 percent of the afflicted birds and morbidity up to 90percent, the significance of this disease has even greater economicimplications. Many chickens which may appear clinically normal aredowngraded during dressing, because of hemorrhages in the musculature ofthe legs, thighs and breast.

In the past, various theories have been advanced as possible causes ofthis elusive disease, but with no conclusive evidence that any of theadvanced theoretical factors has been responsible for the classicalfield syndrome. For example, one of the most popular theories centers onavitaminosis K induced by sul-faquinoxaline toxicity, or by antagonismof this coccidiostat or other drugs to vitamin K. However, the fact thatthe syndrome has occurred in the complete absence of sulfaquinoxaline orother drugs regarded as antimetabolitic to vitamin K precludes thistheory. The avitaminosis K theory is invalidated further by the factthat very little beneficial response was elicited from theadministration of therapeutic quantities of vitamin K to the afflictedbirds. Therefore, methods used in the past for the amelioration of thishemorrhagic disease have not been successful.

against development of toxic fungal metabolic by-products for a longperiod of time under adverse conditions. It is with the prevention ofsuch cereal borne intoxication that the present invention isparticularly concerned. Asused in this specification, the term cerealshall mean any grass yielding grain such as corn, wheat, or rice, alsothe grains so produced; a prepared foodstuff of grain such as oatmeal orbran; the conversion product such as bread, as well as compositionsthereof, and to include fabaceous plants and their products such aspeanuts, soybeans, peanut meal an soybean meal and others.

Once having determined the etiology of the hemorrhagic syndrome andhaving classified it as a mycotoxicoses, I then discovered that theproduction of toxic materials, or at least toxic levels of thesematerials, could be suppressed without the necessity of completelyinhibiting the growth of the fungi which would normally :produce them. Ifurther discovered that this could be accomplished by incorporating8-hydroxyquinoline into the cereal substrate in less than fungicidalconcentrations which will hereinafter be described. These quantities mayvary somewhat with different cereal substrates as well as with differentfungi, but in general are within the range of from about 10'to about 400parts per million based on the normal weight of the cereal. The upperlimits of the range ase relative to certain conditions such as humidity,the substrate and the fungus or fungi involved. Therefore, the upperlimit of the range may best be defined as that concentration of8-hydroxyquinoline which is just below that concentration which iseffective as a fungicide.

It has been found that the occurrence of mycotoxicoses can be preventedby the use of S-hydroxyquinoline in less than fungicidal concentrations.The true mechanism of activity is unknown, however, it is believed thatsmall amounts of the 8-hydroxyquinoline interfere with the metabolism ofthe fungi and thereby prevent the elaboration of toxic by-products.While there may be a proliferation of the fungi in the cereal, asubsequent ingestion of the infected cereal with less than fungicidalcon- 'centrations of 8-hydroxyquinoline does not produce manifestationsof mycotoxicoses.

Therefore, the invention relates to a cereal composition containing acereal which when infected with fungi is normally capable of producingtoxic levels of metabolic by-products in conditions of relatively highhumidity,

I have, discovered that toxic elaborated products as the result ofgrowth of fungi on cereals fed to animals are the basic cause of thishemorrhagic disease. Thus, the hemorrhagic syndrome may be classified asbeing a mycotoxicoses, which is a toxemia produced in animals that haveconsumed toxic substances secreted by fungi. The relationship of thetoxic fungal metabolic products to the hemorrhagic syndrome has beenconfirmed con clusively by production of the hemorrhagic syndrome underlaboratory and field conditions.

Therefore, a principal object of my invention is the prevention of thosemaladies associated with ingestion of cereals, hereinafter defined, suchas straw, hay, grain, and processed feed on which fungi proliferate assaprophytes and give rise to toxic products, either by secretion ofmetabolic products into the substrata, or by the toxlc substance beingpresent as an endotoxin in the mycelium or in the fruiting bodies of thefungus. Because of the possibility that small portions of infectedcereals may stand for a considerable period of time before they areingested, it is necessary that the cereals be protected and aconcentration of S-hydroxyquinoline, which is less than the fungicidalconcentration, but sufiicient to suppress the production of toxic levelsof metabolic byproducts, and thereby prevent the occurrence ofmycotoxicoses.

For the reasons outlined above, a concentration of 10 to 400 ppm. inpoultry feed, for example, does not necessarily suppress profuse fungalproliferation in inoculated feed, however, an ingestion of this feed bychicks does not result in any manifestations of the poultry hemorrhagicsyndrome. Therefore, although there may be profuse fungal proliferationin the inoculated feed, there is sufficient suppression of the poultryhemorrhagic syndrome.

Although the invention is not limited to a particular group of fungi,the following have been observed as being capable of producingmyco-toxicoses. Typical of such fungi are: Aspergzllus clavatus,Aspergillus flavus, Aspergillus chevalieri, Aspergillus fumigatus,Penicillium rubrum, Penicillium purpurogenum, other strains ofPencillium and Alternaria species. Certain Fusarium and Mucoralcs arealso sometimes found. Undoubtedly there exist other toxic fungi whichhave not as yet been isolated and identified. Mycotoxicoses have beensuppressed by the use of. 8-hydroxyquinoline in less than fungicidalconcentrations, in cereals which have been inoculated with suspensionsof the aforementioned fungi.

The following examples are provided for illustrative purposes and mayinclude particular features of the invention. However, the examplesshould not be construed as limiting the invention, many variations ofwhich are possible without departing from the spirit or scope thereof.

Example L-Preparation of animal feed for hemorrhagic syndrome studyEight kilograms of non-sterile broiler mash were weighed and themoisture content of the feed determined by means of a Delmhorst MoistureDetector. To the sample were added 40 ml. of an aqueous suspension of amixture of toxic fungi (Alternaria sp., Aspergillus clavatus,Aspergillus fumigatus, Aspergillus flavus, Aspergillus glaucus,Penicillium rubrum' andtwo unidentified species of Penicillia) andsufficient tap water free of chlorine to bring the total moisturecontent of the feed to approximately 21 percent. The fungi had beenisolated from feed and litter obtained from broiler houses in which thepoultry hemorrhagic syndrome was enzootic. The moistened sample was thenpremixed by hand, rubbed through a inch mesh stainless steel screen andmixed mechanically for 5 minutes in a Hobart feed mixer.

The sample was then divided into two lots, and to a small portion of oneof the lots a small quantity of 8-hydroxyquinoline was added. Afterthoroughly premixing the 8-hydroxyquinoline with a spatula, more feedwas added from the lot from which the small portion was taken, andpremixed by hand until the whole lot was premixed, and this wascontinued until the S-hydroxyquinoline content was 100 p.p.m based onthe weight of the moist feed. The lot containing the S-hydroxyquinoline(Lot 2) was then further mixed mechanically for 5 minutes in the Hobartfeed mixer. Lot 1, which contained no S-hydroxyquinoline, served as acontrol. Both sample lots were then transferred to round fiberboardcontainers, equipped with a gauze-covered opening in the lid forventilation, and incubated at ordinary room temperatures. The samplelots were observed periodically for gross and microscopic proliferationof fungi.

After 14 days, a very heavy fungal growth and formation of fruitingbodies was observed in both sample lots. After 18 days, there was aheavier fungal growth observed and further formation of fruiting bodies.On the 25th day both sample lots were removed and dried for 48 hours inincubators adjusted to approximately 45 C.

Example 2.Determination of the efficacy of using less than fungicidalconcentrations of 8-hydr0xyquin0line During the early stages of thepoultry hemorrhagic syndrome, the affected birds show signs ofdepression, diarrhea which is frequently bloody, paleness of combs andWattles, anorexia and varying morbidity and mortality. Autopsy findingsin such birds reveal hemorrhages and congestion in the musculature ofthe legs, thighs, and breast, and in many other tissues, as well aserosion of the mucosa of the proventriculus and gizzard. The othertissue changes are characteristic of an acute degenerative condition inthe liver and kidneys.

Autopsy findings, in birds which have progressed to the advancedsyndrome, will reveal blood pigments in the liver, proteinaceous andhematogenous casts in the distal convoluted and collected tubules of thekidneys, and paleness, and gelatinization of and depression in theblood-forming elements in the bone marrow.

Two groups of New Hampshire-Red Barred Rock cross, day-old chicks, 20per group, were fed ad libitum respectively, the following lots of feed:the inoculum-containing lot with 100 p.p.m. of S-hydroxyquinoline,hereinafter called Lot 2, and the inoculum-containing lot, without theS-hyroxyquinoline, Lot 1.

On the fourth day of feeding, chicks in the group which were beingmaintained on the feed of Lot 1, showed signs of the early symptoms ofthe hemorrhagic syndrome, that, is depression and fetid diarrhea. Thiscondition prevailed until the 17th day when depression was no longerobserved; however, diarrhea in this group was observed on the 21st day.Thereafter, chicks in this group grossly appeared normal.

The second group of chicks which were maintained on the feed of Lot 2,in which 8-hydroxyquinoline was added, showed none of the early symptomsof the hemorrhagic syndrome. The chicks of this group grossly appearednormal for the full period of the test.

Additional lots of feed were prepared following the procedure set forthhereinabove. At the end of four weeks, the supply of feed was exhausted,and 10 chicks from each group were sacrificed by decapitation, andexamined grossly at autopsy for manifestations of the syndrome.

These test results show that although p.p.m. of 8- hydroxyquinoline doesnot suppress profuse fungal proliferation in the inoculated feed, thereis suppression of the hemorrhagic syndrome. This is shown by theautopsy, as well as by gross observation which revealed a pronouncedsuppression of the hemorrhagic syndrome.

Attempting to achieve greater lucidity, as well as speed, in theinterpretation of the results of the autopsy, an arbitrary hemorrhagicsyndrome score was assigned to the twelve organs examined at autopsy. Anormal organ is given the value zero, slight manifestations 1, moderatemanifestations 2, pronounced manifestations 3. The score is then totaledfor each of the carcasses examined, and a mean score is determined bydividing the total score of all the carcasses examined by the number ofcarcasses. The results of the autopsy of the two groups of chicks areset forth in the following table.

Example 3.C0mparative studies of the eflfcacy of less than fungicidalconcentrations of 8-hydroxyquin0line, and fungicidal concentrations of8-hydroxyquin0line, 0n the suppression of the hemorrhagic syndrome Ninelots of non-sterile broiler mash containing no antibiotic orcoccidiostat were inoculated with a mixture of toxic fungi (Alternariasp., Aspcrgillus clavatus, Aspergillus fumigatus, Aspergillus flavus,Aspergillus glaucus, Paecilomyces varioti, Penicillium purpurogenum,Penicillium rubrum, and three unidentified species of Penicillia),adjusted to 21.8 percent moisture, and containing respectively, 0, 10,50, 100, 200, 250, 300, 400 and 500 ppm. of S-hydroxyquinoline, wereincubated at ordinary room temperatures, observed for fungalproliferation over a period of 13 to 14 days, then dried and fed today-old chicks, 10 animals per group. The chicks, which were maintainedon batteries, had free access to water and the prepared feeds, and wereobserved daily for gross manifestation of toxicity and weighedperiodically until the supply of feed became exhausted in any one group.After 4 weeks the feed supply in one group became exhausted. At thistime, all chicks in each group were sacrificed by decapitation andexamined at autopsy for gross manifestation of poultry hemorrhagicsyndrome.

The degree of fungal proliferation in the feed samples, Production ofthe hemorrhagic syndrome.-Normal Observed during the experiment, appearsin the following boiler mash having a moisture content of 12.8 percent,table. The degree of fungal proliferation is designated as containing noantibiotic or coccidiostat, was inoculated follows: with 0.5 percent ofdry inoculum of the mixture of toxic fungi described in Example 1. Thefeed was. then fed to 5 gig 321 chicks maintained on wood-shavingslitter on a concrete floor in a building having a total area of 100square feet.

profuse growth One hundred chicks, 50 males and 50 females, had free ASindicated in the following table, the lots of e access to the feed inhoppers and water in normal water- Containiflg from 0 to 100 P-P- ofs-hydfoxyqllinolme 0 ing devices located on the litter. Litter and feedsubse- ShOWed Pfefllse fungal Proliferation after 14 Y Slightquentlyscattered in the litter by the chicks, were examined growth Was ein the lots Containing 200-300 P-P- periodically by steriomicroscopicmeans for fungal pro- 0f 8-hydroxyqumohneafter 14 y N fungal growthliferation, and the chicks observed for gross manifestation was observedin the samples contalnmg 400 and 500 f i i p m. of 8-hydroxyqu1nolme. 15Nineteen days after initiation of the experiment, slight fungalproliferation was observed in feed scattered by the TABLE 2 chicks inlitter near water devices, and heavy fungal growth was observed threedays later. Fungal prolifera- Growth on Days Indicated tion graduallybecame more evident in feed scattered in ggl lgrg g i y other parts ofthe chicken house and was pronounced in feed and litter in all parts ofthe house by the fourth 6 8 10 14 week On the 22nd day of theexperiment, slight depression,

1 i 2 i diarrhea, and paling of combs and Wattles were observed 1 1 4 425 in some chicks and became more pronounced in approxig i mately 25percent of the flock at 4 /2 weeks. Death began to occur in chicks at 4weeks and mortality was 10 Days percent at 5 /2 weeks.

Gross autopsy findings were typical of those observed 6 8 n 13 in fieldcases of poultry hemorrhagic syndrome, namely,

superficial and deep hemorrhages and congestion in the 0 0 0 1musculature of the legs, thighs, and breasts; hemorrhages 0 0 0 1 andcongestion in the lungs, liver, spleen, kidneys, small 8 g 8 intestineand caeca, and pronounced erosion in the proventriculus and gizzard. At7 weeks, when the experiment was concluded, moderate depression andfetid diar- It can be conclusively stated, as a result of autopsy Thea eObserved in Practically 100 Percent Survivfindings, that although the8-hydroxyquinoline did not mg l f Flve males and five females sacrificedy suppress fungal proliferation in the lots containing 1040O'decaplfatlon Showed gTQSS P X findlngs of Severe mam ppm. of thecompound, the chicks that consumed these 40 lfestatlons 0f p jyhemorrhagic Syndrome having an lots showed a pronounced suppression ofthe hemorrhagic average hemorfhagle Syndrome re of 30. syndrome. It hasbeen observed in many experiments w 3150 determined that the hemoffhaglcSyndrome that chicks used as controls and which received so-calledeXlSts In two forms, ly e acute d Chronic. The normal feed as it ispurchased on the open market, at aeufe e ePP after ln'gestloll of lafgequantities of autopsy, had an average hemorrhagic score f 4 to 5 ahighly toxic substrate, and is characterized for the most Thus, the meanscores of 5 for p.p.m. level, as well P X a hemorrhflgle dletheslsPloethora, and as those f h 25.0 levels and above can be generativechanges m the llver and kidneys. When small considered to approximatenormal values. It can also be afnounts P feed are admlmtered Over a longPestated that the 8-hydroxyquinoline in the concentrations of tlmeammals develop Venous Stages of Chronic tested had no apparent effect ongain in body weight. 50 tOXICOSeS- The most Outstanding symptom of thefinal The results of the autopsy are set forth in the following tag ofthe chronic syndrome is pronounced depression table, again utilizing thearbitrary hemorrhagic synof the haemophoeitic system. drom score setforth hereinabove. Suppression of the hemorrhagic syndr0me.To con- TABLE3 Concentration of Compound (p.p.m.)

Number of Chicks Autopsied 10 10 10 10 10 10 10 9 10 Total HemorrhagicSyndrome Score 160 89 52 71 55 50 47 26 30 Mean Hemorrhagic SyndromeScore... 16 9 5 7 6 5 5 3 3 Example 4.-Production of the hemorrhagicsyndrome clusively prove the relationship of the toxic fungal metaunderfield conditions bolic by-products to the hemorrhagic syndrome, it is believed that a suppression of the fungal proliferation in feed and litterwill concomitantly result in a suppression of the poultry hemorrhagicsyndrome in chicks main- The relationship of the toxic fungalelaboration products to the hemorrhagic syndrome was confirmed by aneffective production of the hemorrhagic syndrome under field conditions.The relationship was further validated, tflined under field Conditions hwould be substanby concurrently suppressing the hemorrhagic syndrome lfthe Same as those fiescrlbed 111 the foregomg P under field conditions,using the fungicidal concentration ductlon of the hemorrhagic syndrome.

of S-hydroxyquinoline. To the inoculated feed were added by rubbingthrough SO-mesh screen, 500 p.-p.m. of 8-hydroxyquinoline, mixed for 15minutes in a cement mixer and fed to 100 chicks.

Steriomicroscopic examination of litter and feed scattered in litter bythe chicks for 9 weeks did not demonstrate fungal proliferation. Inaddition, during this period the chicks demonstrated no evidence ofpoultry hemorrhagic syndrome. Five males and five female chicks weresacrificed at this time by decapitation. The autopsy findings resultedin showing that the chicks for all practical purposes were normal,having an actual average syndrome score of less than 1.0. This is astriking comparison to the. fact, that as indicated in Tables 1 and 2,the chicks that consumed the feed with the inoculum alone demonstratedan average syndrome score of 16.

I claim:

1. A method of treating cereals to prevent the formation therein, by theaction of fungi in the presence of moisture, of amounts of metabolicby-products that will be toxic to animals ingesting the same, whichcomprises incorporating into said cereal 10-400 ppm. of8-hydroxyquinoline.

2. A cereal, which when infected with fungi is normally capable ofproducing toxic levels of metabolic byproducts in conditions ofrelatively high humidity, con taining 10-400 ppm. of8-l1ydroxyquinoline.

References Cited by the Examiner UNITED STATES PATENTS 2,711,962 6/1955Bickoff 99-8 2,844,508 7/1958 Weiss et a1. l6738 OTHER REFERENCES A.LOUIS MONACELL, Primary Examiner.

D. DONOVAN, Assistant Examiner.

2. A CEREAL, WHICH WHEN INFECTED WITH FUNGI IS NORMALLY CAPABLE OFPRODUCING TOXIC LEVELS OF METABOLIC BYPRODUCTS IN CONDITIONS OFRELATIVELY HIGH HUMIDITY, CONTAINING 10-400 P.P.M. OF8-HYDROXYQUINOLINE.